KR101229436B1 - Bone regeneration material and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a bone regeneration material and a method of manufacturing the same applied to the bone damage site. According to the present invention, in the field of orthopedic, dental, neurosurgery and the like to perform treatment using demineralized bone substrate for the purpose of reconstructing damaged bone, the implantability is excellent so that the procedure is easy and does not flow down in vivo so as not to flow after transplantation. Physical features that can maintain form are very important. In addition, it is also possible to provide a material for transplantation using a demineralized bone substrate to exhibit excellent bone regeneration ability without causing rejection in the transplanted living body.

Description

Bone regeneration material and manufacturing method thereof

The present invention relates to a bone regeneration material and a method of manufacturing the same applied to the bone damage site. More specifically, it relates to a bone regeneration material containing gelatin crosslinked with radiation.

As the development of medical technology and the increase of accidents, disasters and dental treatments, the importance of biomaterials is increasing. Biomaterials refer to materials used to treat, enhance, replace, or restore the function of human tissues or organs in the human body for a short or long period of time as artificial, natural, or composites thereof, except pharmaceuticals.

In the field of treatment of damaged bone tissue, a variety of implants are known whose main purpose is their reconstruction. These implants are used for a wide variety of purposes, including the treatment of nonunion fractures, joint fixation, filling of the bone cavity, replacement of bone and joint loss, strengthening of the tibia and skull, and fusion of growth plate cartilage.

Especially in the field of bone reconstruction through bone graft, autograft is the most commonly used method of taking a part of bone and transplanting it into the graft (S. Stevenson, Biology of bone graft, Orthop Clin North Am, 30). , 543 (1999). The advantage of autografts is that bone formation can occur more smoothly than other implants by providing live cells that act not only on the bone matrix but also on bone formation. Therefore, the halving and the probability of failure of the treatment effect by the immune rejection reaction can be greatly reduced. In addition, since there is little bone absorption, fewer fatigue fractures, and excellent bone induction and bone conduction ability, the union and thickening of the bone proceed quickly, which is advantageous for the reconstruction of large bone loss. However, in spite of the advantages of such autograft, since autograft has to collect bone from the body of the subject, there is a disadvantage that there is always a problem of secondary infection, which is necessarily accompanied by the pain of the patient and the surgical operation.

In order to solve this disadvantage, an alternative demineralized bone matrix (DBM) is proposed. The demineralized bone matrix, known to have excellent osteoinductive capacity, or the demineralized bone matrix, was first introduced in 1889 by Senn as a carrier for injecting preservatives into the human body of patients with osteomyelitis. Subsequently, the demineralized bone matrix was substantially visualized for clinical purposes only after the induction of bone formation by the demineralized lyophilized bone through Urist's study. Since then, many studies have been conducted on demineralized bone matrix, and it has been reported that, in Mulliken and Glowacki, demineralized bone is more effective in inducing bone formation than non-mineralized bone.

The demineralized bone matrix has the advantage of not only the ability to induce bone but also additional surgery for bone extraction. However, demineralized bone substrates are inferior in affinity with aqueous solutions, and have a disadvantage of difficulty in maintaining shape when transplanted alone. In addition, when not used with a special stabilizer or carrier, there is always a possibility that bone morphogenetic proteins (BMPs) contained in the demineralized bone matrix can be easily degraded in vivo. When demineralized bone matrix itself is not possible transplantation exists.

Therefore, in the field of orthopedic, dental, neurosurgery, etc., which uses demineralized bone substrates for the purpose of reconstructing damaged bones, it has excellent injectability to facilitate the procedure and maintains its shape in vivo so that it does not flow down after transplantation. The physical features that are possible are very important. In addition, it is also a requirement of the material for transplantation using demineralized bone substrate to be able to exhibit excellent bone regeneration ability without causing rejection in the transplanted living body.

In the manufacturing process of the graft material using the demineralized bone substrate, the deliming process and the sterilization process are involved. In the deliming and sterilization process, there is a need to develop products and processes with higher reliability by minimizing the use of chemicals to prevent contamination by toxic components while completely eliminating pathogens.

Under such technical background, the present inventors have made intensive efforts to develop a bone regeneration material having better bone regeneration ability, biocompatibility, and shape maintaining ability, and have completed the present invention.

After all, the technical problem to be achieved by the present invention is to provide a bone regeneration material having excellent bone regeneration ability, biocompatibility, flexibility, shape maintaining ability.

Another technical problem to be achieved by the present invention is to provide a method for producing the bone regeneration material.

Another technical problem to be achieved by the present invention is to provide a bone regeneration sheet comprising the bone regeneration material.

According to an aspect of the present invention, a bone regeneration material including 40 to 90% by weight of bone powder and 10 to 20% by weight of gelatin may be provided.

According to an embodiment, the bone powder may be selected from the group consisting of cortical bone, demineralized bone matrix (DBM), and mixtures thereof.

According to one embodiment, the bone powder may be crushed to a size of 0.01 ~ 2mm.

According to one embodiment, the bone meal may be of human origin.

According to one embodiment, the gelatin may be of human origin.

According to one embodiment, the gelatin may be cross-linked through radiation treatment.

According to one embodiment, the radiation may be selected from the group consisting of gamma rays, electron beams (E-beam) and X-rays.

According to one embodiment, the absorbed dose of the radiation may be 10 ~ 30KGy.

According to one embodiment, the moisturizing agent may further comprise 0 to 50% by weight.

According to one embodiment, the moisturizing agent may be glycerol.

According to another aspect of the invention, the step of demineralizing the cortical bone with HCl and crushed to prepare a bone powder (bone powder); Hydrolyzing the bone meal with a calcium hydroxide (Ca (OH) ₂ ) solution; Extracting gelatin from the hydrolyzed bone meal; Mixing the extracted gelatin with bone meal; And irradiating the mixture of gelatin and bone meal with radiation may be provided.

According to an embodiment, the bone powder may be selected from the group consisting of cortical bone, demineralized bone matrix (DBM), and mixtures thereof.

According to one embodiment, the step of hydrolyzing the bone meal with calcium hydroxide solution is 0.1 to 2% by volume of calcium hydroxide solution at a ratio of 15 to 25ml per 1g bone meal It can process for 30 to 70 days at 10-20 degreeC.

According to one embodiment, the step of extracting gelatin from the demineralized and hydrolyzed bone meal may be made by suspending the demineralized bone meal in distilled water or saline and filtering while increasing the temperature from 50 ℃ to 90 ℃.

According to one embodiment, the step of mixing the gelatin with bone meal may be to mix the bone powder 40 ~ 90% by weight, gelatin 10 ~ 20% by weight in the total mixture.

According to one embodiment, the step of irradiating the mixture of gelatin and bone meal may be to irradiate the radiation so that the absorbed dose is 10 ~ 30KGy.

According to another aspect of the present invention, the bone regeneration sheet is attached to one or both sides of the bone regeneration material (Sheet).

According to one embodiment, the sheet may be at least one selected from the group consisting of epithelium, endothelium and connective tissue.

According to an embodiment, the sheet may include an extracellular matrix or a pericardium derived from a mammal.

According to one embodiment, the sheet may include collagen or a basement membrane derived from a mammal.

According to the present invention, in the field of orthopedic, dental, neurosurgery and the like to perform treatment using demineralized bone substrate for the purpose of reconstructing damaged bone, the implantability is excellent so that the procedure is easy and does not flow down in vivo so as not to flow after transplantation. Physical features that can maintain form are very important. In addition, it is also possible to provide a material for transplantation using a demineralized bone substrate to exhibit excellent bone regeneration ability without causing rejection in the transplanted living body.

1 is a result of the flexibility test for bone graft material of the present invention.
Figure 2 (a) is a picture of the lumbar spine exposed L4 ~ 5 after the cut off both muscles around the spine of the SD rat.
Figure 2 (b) is the bone regeneration material according to the invention inserted into the lumbar spine L4 ~ L5 of the SD rat.
3 is an X-ray photograph taken immediately after the procedure. (A) is a control group, (B) is a bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only a cortical bone component, (D) The bone graft material was transplanted by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.
Figure 4 shows the X-ray imaging results of the bone graft taken 6 weeks after transplantation. (A) is a control group, (B) is a bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only a cortical bone component, (D) The bone graft material was transplanted by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.
Figure 5 shows the micro CT results of the bone graft taken 6 weeks after transplantation. (A) is a control group, (B) is a bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only a cortical bone component, (D) The bone graft material was transplanted by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.
Figure 6 shows the results of H & E histology of bone graft taken 6 weeks after transplantation. (A) is a control group, (B) is a bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only a cortical bone component, (D) The bone graft material was transplanted by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.
Figure 7 shows the X-ray results of the bone graft taken 12 weeks after transplantation. (A) is a control group, (B) is a bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only a cortical bone component, (D) The bone graft material was transplanted by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.
Figure 8 shows the micro CT results of the bone graft taken 12 weeks after transplantation. (A) is a control group, (B) is a bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only a cortical bone component, (D) The bone graft material was transplanted by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.
9 shows the results of H & E histology of the bone graft taken 12 weeks after transplantation. (A) is a control group, (B) is a bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only a cortical bone component, (D) The bone graft material was transplanted by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.
FIG. 10 is a MT stained picture after bonding a sheet (SureDerm) including a gelatin-containing bone graft material and a miracle membrane.
Figure 11 is a four-week histological examination after implantation of gelatin-containing bone graft material and the acellular dermal layer conjugated subcutaneously in rats. M denotes muscle, S denotes SureDerm, D denotes dermis, DBM denotes bone regeneration, and arrow denotes basement membrane part.

Hereinafter, the present invention will be described in more detail.

An object of the present invention is to provide a bone regeneration material having excellent bone regeneration ability, biocompatibility, flexibility and shape maintenance ability and a method for producing the same.

According to an aspect of the present invention, bone regeneration material comprising 40 to 90% by weight of bone powder, 10 to 20% by weight of gelatin cross-linked by radiation may be provided.

In this case, the bone powder may be any one selected from the group consisting of cortical bone, demineralized bone matrix (DBM), and mixtures thereof.

In addition, the bone that is the raw material of the bone meal is available from ESB, TSC, Hopital Erasme, TBI, Allosource and Lifelink, etc., Rt / Lt. Humerus (left and right humerus), Rt / Lt. Radius, Rt / Lt. Ulna (vertebrae), Rt / Lt. Femur (femur), Rt / Lt. Tibia (Tibia), Rt / Lt. Fibula can be used, but not limited thereto, and any kind of human bone or animal bone may be used.

The bone powder refers to a state in which the bone is crushed to have a particle size of 0.01 ~ 2 mm. More preferably, the size of the crushed bone particles is preferably 0.15mm ~ 0.85mm.

The cortical bone is called the dense bone as part of the cortex, which is the outer part of the bone that is responsible for supporting the body's main functions such as bone support, protection of internal organs, transmission of exercise power, and the storage and release of various chemicals, especially calcium. It is a very dense bone tissue.

The origin of the cortical bone used in the present invention may be derived from a different species from the object to be treated, but in the same species, higher bone regeneration effects and morphological maintenance after bone regeneration can be expected. When it is intended to be treated in humans, it is preferable that the cortical bone derived from the human body. Similarly, the gelatin is also irrelevant to the object to be treated and is derived from a different species (species), but the same species can be expected to have a higher bone regeneration effect and the effect of maintaining the shape after bone regeneration. In the case where the procedure is intended for humans, gelatin derived from the human body is preferable.

According to one embodiment of the invention, the gelatin may be cross-linked with radiation, the radiation may be selected from the group consisting of gamma rays, electron beams (E-beam) and X-rays. More preferably, they may be electron beams and gamma rays.

According to one embodiment, the absorbed dose of the radiation is 10 ~ 30KGy, more preferably 15 ~ 25KGy. If the radiation absorbed dose is less than 10KGy, the crosslinking of the gelatin does not proceed sufficiently, so that the tensile strength of the final bone regeneration material is lowered, and thus the flexibility required for transplantation is also worsened, which is not preferable. When the radiation absorbed dose is 30KGy or more, deformation of the biomolecules in the composition for bone regeneration and excessive generation of radicals may be caused, which is not preferable.

According to the present invention, a moisturizing agent may be further included in the mixture of gelatin crosslinked with bone meal and radiation.

According to one embodiment, the moisturizing agent may not be added as an optional component, but when added, it may be glycerol and 0 to 50% by weight of the total bone regeneration material may be added. When the moisturizing agent is added in excess of 50% by weight, the strength and ductility of the bone regeneration material is low, even if the cross-linking process may be difficult to maintain flexibility and shape is not preferable.

According to another aspect of the present invention, the bone regeneration material may be put into a mold of a desired shape and subjected to a molding process at a low temperature.

Regardless of the form, the bone regeneration material does not necessarily have to match the shape of the bone injury to be transplanted, and it is sufficient if the shape is processed to a similar shape. However, if necessary, the shape of the bone injury site of the transplantation target may be grasped in advance, and a shape suitable for the implant may be prepared, and the shape of the membrane or the structure of the band may be preformed so as to be generally applicable to various situations.

To this end, various materials for increasing the moldability of the bone regeneration material may be further added. That is, linear materials, tubular materials, particulate materials, and amorphous materials, which are excellent in biocompatibility, may be further added to improve physical properties of the bone regeneration material. This type of material can be selected from materials such as collagen, CMC (Carboxymethylcellulose) or animal-derived gelatin, and there is no particular limitation as long as it does not cause an immune response to the transplant target.

According to another aspect of the invention, the step of demineralizing the cortical bone with HCl and crushed to prepare a bone powder (bone powder); Hydrolyzing the bone meal with a calcium hydroxide (Ca (OH) ₂ ) solution; Extracting gelatin from the hydrolyzed bone meal; Mixing the extracted gelatin with bone meal; And irradiating the mixture of gelatin and bone meal with radiation may be provided.

In the deliming step, cortical bone is first delimed for 2 to 8 hours by adding 0.1 to 2N of HCl. Unlike this, the demineralized bone matrix (DBM), which has been subjected to the demineralization process as described above, may be directly introduced into the next process without further demineralization. There is no limit to the number of deliming treatments, but it is sufficient that calcium is no longer extracted even in the treatment of HCl.

The demineralized cortical bone (DBM) is treated with calcium hydroxide solution for 30-80 days for hydrolysis of collagen. It is desirable to stir at least once daily during the treatment and to change fresh calcium hydroxide solution every week.

At this time, the concentration of the calcium hydroxide solution for hydrolysis is preferably 0.1 to 2% by volume. At a concentration of less than 0.1 vol%, the hydrolysis of collagen does not occur smoothly in the bone, and at a concentration of 2 vol% or more, it is not preferable because residual calcium hydroxide is difficult to remove after the hydrolysis process due to the accumulation of calcium hydroxide.

The calcium hydroxide solution is preferably treated at a ratio of 15-25 ml per 1 g of bone flour, but less than 15 ml may not sufficiently undergo hydrolysis treatment, and the ratio of bone flour at suspension is so high that too many filtration processes for collecting gelatin are required. It is not preferable because it may take time, and if it exceeds 25ml it is not preferable because the later removal process of calcium hydroxide becomes unnecessarily long.

The hydrolysis treatment is preferably treated for 30 to 70 days at 10 ~ 20 ℃ calcium bone solution in the calcium hydroxide solution, it can be carried out through immersion and stirring (Agitation) as necessary. At this time, when the treatment temperature is less than 10 ℃, demineralization reaction between calcium hydroxide solution and bone flour may be inhibited due to coagulation of lipid and protein components in bone flour, which is undesirable. The increased risk of degradation and contamination by microorganisms is undesirable. In addition, when the treatment process is less than 30 days is not sufficient hydrolysis, and if more than 70 days it is also undesirable to increase the possibility of excessive hydrolysis and contamination by microorganisms.

The demineralized and hydrolyzed bone meal is preferably washed with PBS and distilled water, and the pH is adjusted to neutral to enter the gelatin extraction operation immediately.

According to one embodiment of the present invention, the step of extracting gelatin from the demineralized and hydrolyzed bone meal is a gel supernatant produced by suspending the demineralized bone meal in distilled water or saline and filtering it. It may be made by drying.

At this time, it is preferable to add a distilled water or physiological saline of 5 to 15ml per 1g of bone meal to prepare a suspension, and to extract the extract every 10 ° C. while gradually increasing the temperature from 50 ° C. to 90 ° C. In this case, when the temperature is lower than 50 ℃ gelatin component may be agglomerated and not easy to extract, it is not preferable, and if the temperature is increased above 90 ℃ geological modification may occur is not preferable.

At this time, the gelatin extraction time for each temperature is preferably 4 to 6 hours, if less than 4 hours gelatin is not properly eluted extraction efficiency can be lowered, if exceeding 6 hours the process is too long Not preferred.

The suspension collected through the above process is preferably filtered through a filter paper to remove the particles of bone meal and collect only the solution. Alternatively, it is also possible to precipitate the bone meal by centrifugation and the like and collect only the supernatant.

The collected supernatant is preferably concentrated to 50 ~ 70 ℃ in a vacuum concentrator. Through this gelatin is concentrated to a gel state, and dried using a dryer or the like to obtain a solid gelatin. Grind this to a size of 0.5 ~ 2mm with a grinder. At this time, when the particle size is less than 0.5mm, it is not preferable because it will be agglomerated when dissolving gelatin in the future.

According to one embodiment of the present invention, the step of mixing the gelatin with bone powder is preferably mixed so that the bone powder in the total mixture is 40 to 90% by weight, gelatin 10 to 20% by weight. In this case, if the bone powder is less than 40% by weight is not preferable because the bone regeneration promoting effect of the final product is not preferable, and if it exceeds 90% by weight is not preferable because the flexibility of the product is lowered.

If the gelatin is less than 10% by weight, it is not preferable because the flexibility (tensile strength) of the resulting bone regeneration material is lowered, and when it exceeds 20% by weight, the moldability is lowered, which is not preferable.

According to the present invention, the mixture of gelatin and bone meal can be irradiated. In order to irradiate the radiation, the mixture of gelatin and bone meal may be put in a mold for molding to stabilize at low temperature, and then separated and transferred to a container for radiation transmission and packaging, followed by irradiation. In this step of irradiating the radiation it is preferable that the absorbed dose of radiation is 10 ~ 30KGy, more preferably 15 ~ 25KGy.

In this case, when the radiation absorbed dose is less than 10KGy, the crosslinking of the gelatin may not proceed sufficiently, so that the tensile strength of the bone regeneration material may be lowered, and thus the flexibility required for transplantation may be deteriorated, which is not preferable.

The bone graft material prepared according to the above embodiment may be treated with various media depending on the area to be treated and the form and extent of the injury. When the bone graft material is treated, the soft tissue is regenerated earlier than the bone tissue at the injured portion of the bone tissue, and the phenomenon may rise. In this case, it is difficult to expect normal bone tissue regeneration.

Therefore, the bone regeneration material according to the present invention can prevent this phenomenon by applying a structure that can shield the soft tissue during the procedure to the bone graft site.

In another aspect according to the present invention, in order to solve this problem, the bone regeneration material may be attached to one side of the sheet (Sheet) to provide a sheet for bone regeneration that can prevent the soft tissue from invading the bone graft site.

In addition, when the bone graft is attached to the bone regeneration material, the bone graft material is attached to the sheet to prevent the regeneration of the bone regeneration material. This is more advantageous for bone induction because the bone regeneration material can be more stably fixed to the bone defects.

At this time, the sheet may be skin tissue of mammals, the epidermis is removed for the body procedure, cells of the dermal layer may be removed to remove immunogenicity.

According to one embodiment, the skin tissue may be a basement membrane (basement membrane).

In the present embodiment, the mammalian base membrane is used for the shielding of the bone graft site, but there is no side effect on the living body, and any medium capable of preventing the invasion of the soft tissue to the bone graft site may be any of the bone regeneration materials according to the present invention. Can be used together, for example, collagen membrane, mammalian-derived pericardium (Pericardium) and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

In the preparation of bone regeneration material, it contains the gelatin derived from the human body to increase biocompatibility, and crosslinking and sterilizing it with radiation to provide sufficient sterilization effect on pathogens, as well as tensile strength suitable for maintaining shape before and after the procedure. It was. The bone regeneration material that has undergone the radiation crosslinking process has excellent flexibility and can be easily processed into a form suitable for the procedure. It could be transformed. In one embodiment of the present invention was prepared in the form of a strip (strip) substrate that is a simple procedure for the damage to the vertebrae.

Pretreatment of raw materials and Demineralized bone matrix (DBM)  Ready

The raw bone is Rt / Lt. Obtained from ESB, TSC, Hopital Erasme, TBI, Allosource and Lifelink. Humerus (left and right humerus), Rt / Lt. Radius, Rt / Lt. Ulna (vertebrae), Rt / Lt. Femur (femur), Rt / Lt. Tibia (Tibia), Rt / Lt. Fibula was used. First, the bones stored in the cryogenic freezer were removed aseptically from the soft tissues attached to the bones. Bones from which soft tissues were removed were placed in ether and degreased. After degreasing, the dried bone was cut and placed in a bone grinder, and the bone was crushed, classified by size, and demineralized with HCl. The demineralized bone meal, ie demineralized bone matrix (DBM), was treated in a supersaturated calcium hydroxide (Ca (OH ₂ )) solution at 15 ° C. for 50 days. At this time, the mixture was stirred once a day and replaced with fresh supersaturated calcium hydroxide solution every 7 days. After the treatment, the demineralized and hydrolyzed demineralized bone matrix powder was washed with PBS and distilled water, and the pH was adjusted to neutral.

Extraction and Filtration of Human-Derived Gelatin

10 ml of distilled water was added per 1 g of the demineralized bone matrix powder prepared in the same manner as above. This was extracted 5 times at 10 ° C intervals up to 50 ~ 90 ° C. The extract was treated for 4 to 6 hours at each temperature step and filtered using a 30 μm filter paper (Whatman). This removed only the demineralized bone matrix particles and collected the remaining solution.

Concentration, drying and grinding of gelatin

Only the demineralized bone matrix particles were removed and the remaining solution was concentrated at 60 with a vacuum concentrator. When the solution is concentrated in a gel state, gelatin remains as a main component, which is dried in a dryer, and then pulverized with a grinder so that the particles become 0.5 to 2 mm.

Bone regeneration  Produce

The gelatinous powder obtained in the above process was dissolved in physiological saline, and then mixed with glycerol as a moisturizer. Since the mixing of glycerol is intended to moisturize, except that the mixing of glycerol is not necessary during the procedure, a composition was prepared.

By applying various composition ratios were prepared bone regeneration material as shown in Table 1.

Control group Example 1 (b) Example 2 (C) Example 3 (d) Demineralized bone matrix (DBM) - 50 gram - 15 gram Cortical bone - - 50 gram 35 gram Gelatin solution
(20% gelatin in saline) 50 cc 50 cc 50 cc 50 cc

The gelatin solution in Table 1 is prepared by mixing 15 cc of glycerol, 35 cc of 0.9% saline solution and 10 g of gelatin.

The control group means that no bone component is included, and Examples 1 to 3 each contain a mixture prepared of demineralized bone matrix, cortical bone, and a combination thereof (30% demineralized bone matrix; 70% cortical bone) in a substrate-shaped mold. It means a bone graft material prepared by stabilizing for 30 minutes at a low temperature of about ℃. The stabilized bone graft material was separated from the mold, transferred to a packaging container, and gamma-ray irradiated with 20KGy to crosslink the gelatin at the same time to investigate its physical properties.

Test Example  One. Bone regeneration  Flexibility testing

The flexibility of each of the bone rejuvenates prepared according to the method was tested. Figure 1 shows a photo of applying a deformation to the bone regeneration material produced in accordance with Example 3 of the present invention. It shows that it is well preserved in its original form without being torn or deformed even in deformation such as strong warping (see Fig. 1 (a) and (b)). Even when the test was left unattended, good flexibility was shown (see FIG. 1 (c)).

Bone regeneration  transplantation

Animal tests were performed to confirm the bone regeneration effect of the various compositions described in Table 1 to test the stability and bone regeneration effect of the bone regeneration material. Animals were used as SD rats ( Sprague-Dawley rat, female, female, 200gram).

First, zoletil50 (virbac korea) was anesthetized by intraperitoneal injection and intramuscular injection in 0.4mg / kg and then fixed on the operating table. The area around the site to be treated was depilated and disinfected using a depilatory cream and a razor. Using a scalpel, cut the skin at the site of the procedure to expose the muscles, and then use a scalpel to cut both muscles around the spine, and then expose L4 to lumbar spine 5 times (see Fig. 2A). , L4, L5 cortical bone (grind) of the lumbar surface was ground. The bone regeneration material was raised and disinfected in L4 and L5 lumbar region, sutured muscle and skin were incised and antibiotics were administered.

Figure 2 (b) is the bone regeneration material according to the present invention inserted into the lumbar L4 ~ L5 of the SD rat before suture.

In the following, X-ray, MicroCT and H & E biopsy were performed immediately after the procedure, and 6 and 12 weeks after the procedure, and the shape and volume maintenance, inflammatory response and bone regeneration-promoting effects of bone grafts according to the present invention according to the transplantation period. Was evaluated.

Test Example  2. Immediately after transplant Bone graft  Insertion aspect confirmation

3 is an X-ray photograph taken immediately after the procedure. (A) is a control that does not contain any bone components, (B) is a transplant of bone graft material containing only demineralized bone matrix components, (C) is a bone graft material containing only the cortical bone components It is appearance. (D) is a transplant of bone graft material prepared by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.

Referring to Figure 3, in the case of (b) because it contains only demineralized bone matrix components, there is almost no mineral component in the bone graft material does not appear clearly even by X-ray, but it was confirmed that the bone graft material is well inserted next to the spine, (c) In the case of (D) and (D), the mineral component was well observed in the X-ray.

Test Example  3. Six weeks after transplantation Bone graft  Shape, volume, inflammatory response Regeneration  Promotion effect

4, 5 and 6 show the results of X-ray, Micro CT and H & E histology of bone graft taken 6 weeks after transplantation.

The X-ray results of FIG. 4 show that there is no significant change in the shape of the transplanted bone graft after 6 weeks. Figure 5 shows the results of the microCT, through which the volume of bone graft material 6 weeks after transplantation is shown in Table 2 by the numerical value.

Implants Right and left Implants  volume
( mm ³ ) Volume total
( mm ³ ) Shape maintenance degree
(Initial volume 360 mm ³ ) Control group L 0 0 0 % R 0 Example 1 (b)
(Contains only DBM ingredients) L 128.79 226.84 63.01% R 98.05 Example 2 (C)
(Contain only Cortical ingredients) L 97.14 219.87 61.07% R 122.73 Example 3 (d)
(DBM 30% + Cortical 70%) L 155.18 306.41 85.11% R 151.23

As shown in Table 3, the volume of bone graft material at 6 weeks after transplantation was higher than 85% of the original volume in the case of bone graft material (c) mixed with demineralized bone matrix and cortical bone.

Figure 6 shows the results of H & E (Hematoxylin and Eosin) staining 6 weeks after transplantation of bone graft material. In the control group (a), common muscle fibers. Focal lymphocyte infiltration has been observed. A small number was observed. In addition, the bone graft material and the surrounding fibrotic connective tissue (Fibrotic connective tissue) is observed as an empty space not connected, and shows high stability and biocompatibility without immune rejection reactions such as inflammatory reactions.

As a result, it can be seen that the bone graft material according to the present invention is excellent in form, volume, inflammatory response and bone regeneration promoting effect even after 6 weeks after transplantation.

Test Example  4. 12 weeks after transplantation Bone graft  Shape, volume, inflammatory response Bone regeneration  effect

7 is an X-ray photograph taken 12 weeks after the procedure. Again (A) is a control, (B) is a transplant of bone graft material containing only demineralized bone matrix components, (C) is a transplant of bone graft material containing only the cortical bone components. (D) is a transplant of bone graft material prepared by mixing the ratio of demineralized bone matrix and cortical bone in a 3: 7 ratio.

Referring to FIG. 7, although the size of the implanted bone graft material was slightly reduced compared to FIG. 4 after 12 weeks after implantation, the shape and location of the bone graft were maintained as it is. MicroCT was performed as shown in FIG. 8 to more accurately confirm the results. The volume of bone graft material was quantified 12 weeks after transplantation through the results of the microCT of FIG. 8.

Implants Right and left Implants  volume
( mm ³ ) Volume total
( mm ³ ) Shape maintenance degree
(Initial volume 360 mm ³ ) Control group L 0 0 0 % R 0 (I)
(Contains only DBM ingredients) L 78.83 152.14 42.26% R 73.31 (All)
(Contain only Cortical ingredients) L 49.58 154.41 42.89% R 104.83 (la)
(DBM 30% + Cortical 70%) L 139.55 266.74 74.09% R 127.19

As shown in Table 3, the volume of bone graft material at 12 weeks after transplantation was maintained at about 40% or more in bone graft material (B) and cortical bone material (B) prepared using only demineralized bone matrix. The bone graft material (C) mixed with demineralized bone matrix and cortical bone was higher than 75% of the original volume.

Figure 9 shows the results of H & E (Hematoxylin and Eosin) staining 6 weeks after transplantation of bone graft material. In the control group (A), only general muscle fibers and muscle structures were observed, and in (B), fibroblasts and osteoclasts were observed around the bone graft material, and bone formation was not observed in the filling area.

In (c), minor lymphocyte infiltration, fibroblast and fibrotic connective tissue, and macrophage infiltration were observed. Was observed.

In (D), fibroblasts and fibrous connective tissue were observed, and a small number of neutrophils and macrophage infiltration were observed.

Through this, it can be seen that the bone graft material of the present invention does not cause an immune rejection reaction, and exhibits high safety and biocompatibility.

Example  4. For bone regeneration  Manufacture of sheets

After heating the bone graft material prepared according to Example 3 to 60 ℃, according to Example 1 of Korean Patent No. 0469661 to remove the cells of the epidermal layer and the dermal layer without damaging the basement membrane to include a base membrane prepared to prevent the immune reaction occurs Placed on the sheet. It was left at room temperature to allow gelatin to seep into the sheet containing the basement membrane. After a certain period of time, the gelatine hardened, and the gelatin in the bone graft material and the gelatin in the sheet containing the basement membrane were coagulated so that the two materials were joined.

FIG. 10 shows a MT stained image after bonding a sheet (SureDerm) including a gelatin-containing bone graft material and a miracle membrane. In MT staining, collagen is bluish and gelatin is reddish. The cell-free dermal layer is mostly composed of collagen, which is bluish. However, because the gelatin is infiltrated and bonded, the gelatin-infiltrated portion appears red.

Example  5. For bone regeneration  Sheet treatment

The bone regeneration sheet prepared according to Example 4 was operated so that the soft tissue adjacent to the injury site did not invade the regeneration range of the bone tissue.

FIG. 11 shows a four-week histology picture after implanting a gelatin-containing bone graft material and a cell-derived dermal layer into the subcutaneous rat. In Figure 11, M is muscle, S is sheet (SureDerm), D is the dermis, DBM represents bone regeneration material, the arrow represents the basement membrane part.

Referring to this, the bone graft material is well held in the subcutaneous shape, it can be confirmed that the invasion of the soft tissue in the portion where the acellular dermal layer and the muscle abutment attached through the above method is prevented.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (20)

A bone regeneration material comprising 40 to 90% by weight of bone powder mixed with human demineralized bone matrix and cortical bone and 10 to 20% by weight of gelatin, and treated with radiation.
delete The bone regeneration material of claim 1, wherein the bone powder is crushed to a size of 0.01 to 2 mm.
delete According to claim 1, wherein the gelatin bone regeneration material, characterized in that derived from human (human).
The bone regeneration material according to claim 1, wherein the gelatin is cross-linked through radiation treatment.
According to claim 1, The radiation is bone regeneration material, characterized in that at least one selected from the group consisting of gamma rays, electron beams (E-beam) and X-rays.
The bone regeneration material according to claim 1, wherein the absorbed dose of radiation is 10-30KGy.
The moisturizer of claim 1 A bone regeneration material, characterized in that it further comprises 0 to 50% by weight.
The bone regeneration material according to claim 9, wherein the moisturizing agent is glycerol.
Method for producing a bone regeneration material comprising the following steps:
Demineralizing the cortical bone with HCl, crushing and mixing with the demineralized bone matrix to prepare bone powder;
Hydrolysis at 10-20 ° C. for 30-70 days at a ratio of 15-25 mL of 0.1-2% by volume calcium hydroxide (Ca (OH) ₂ ) solution per 1 g of bone meal;
Preparing a suspension by adding 5-15 mL of distilled water or physiological saline per 1 g of the hydrolyzed bone meal;
Collecting the extract while raising the temperature of the prepared suspension to 50 ~ 90 ℃;
Extracting gelatin by removing the bone powder and concentrating the collected extract by filtration or centrifugation;
Mixing the extracted gelatin with bone meal; And
Irradiating the mixture of gelatin and bone meal with radiation.
delete delete delete 12. The method of claim 11, wherein the mixing of the gelatin with bone flour comprises mixing 40-90 wt% of bone flour and 10-20 wt% of gelatin in the entire mixture.
12. The method of claim 11, wherein the irradiating the mixture of gelatin and bone meal is irradiated with radiation such that the absorbed dose is 10-30KGy.
A bone regeneration sheet, wherein the bone regeneration material according to any one of claims 1, 3, and 5 to 10 is attached to one side or both sides of a sheet.
The sheet for bone regeneration according to claim 17, wherein the sheet is at least one selected from the group consisting of epithelium, endothelium, and connective tissue.
18. The bone regeneration sheet according to claim 17, wherein the sheet includes an extracellular matrix or a pericardium derived from a mammal.
The sheet for bone regeneration according to claim 17, wherein the sheet includes a mammalian-derived collagen or a basement membrane.

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